Lanthanide complexes preparation and uses thereof

ABSTRACT

The invention relates to compounds, to the complexes they form with a lanthanide, and to the use of the complexes for fluorescence marking or NMR imaging. 
     The complex consists of an Ln ion and a ligand R 2 —C(X—R 1 )(R 3 )—NR 4 R 5 . R 1  is a functional group, X is a single bond or a hydrocarbon-based chain consisting of at least one alkylene or alkenylene group optionally comprising at least one hetero atom or an arylene. R 2  is an anionic group A 2  or a C 1 -C 4  alkylene or alkenylene group bearing at least one such group A 2  and optionally comprising at least one hetero atom. R 3  is H or a C 1 -C 5  alkylene or alkenylene group optionally containing at least one hetero atom, and optionally bearing at least one anionic group A 3 . R 4  is a substituent with light-absorbing properties that forms chelate rings with Ln. R 5  is a substituent that forms chelate rings with Ln.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to compounds capable of forming complexeswith lanthanides, to the complexes obtained and to the uses thereof.

2. Description of the Related Art

Radioactive markers have been widely used in the field of medicalimaging and immunology. On account of the drawbacks of these markers,they have largely been replaced with fluorescent markers.

However, the use of fluorescent markers presents a number of drawbacks,especially due to the auto-fluorescence of the biological media studiedand to the scattering of light in the machines. Lanthanide ion complexeshave been proposed to allow a time-resolved acquisition that eliminatesthese drawbacks. In order to be used as a time-resolved luminescentmarker, a lanthanide ion complex must have numerous characteristics, themost important of which are hydrophilicity, stability in water, thepresence of chromophores capable of generating the antenna effect(Sabbatini, N. et al. Coord. Chem. Rev. 1990, 123, 201), goodphotophysical properties (high absorption, excitation over a readilyaccessible energy range, long lifetime of the excited state and highluminescence quantum yield) and a reactive function that allows covalentgrafting.

The compounds currently proposed rarely combine all of these criteria.For example, the first complexes developed by the company Wallac Oyunder the name Delfia Chelate (Hemmilä, I. et al. Anal. Biochem. 1984,137, 335) do not have good photophysical properties and it is necessaryto perform a lanthanide extraction step in order to measure itsluminescence. The compounds developed by CIS Bio International arecryptates, which require the use of fluoride anions in order to increasethe luminescence (Hemmila, I. et al. Drug Discovery Today, 1997, 2,373). The stability of the compounds also poses serious problems. Thus,the compounds developed by CyberFluor under the name BCPDA only formstable luminescent complexes at high concentrations (Marriott, G. etal., Biophysical Journal, 1994, 67, 957).

Lanthanide complexes, especially of gadolinium, have been used asrelaxation agents or contrast agents for NMR medical imaging (Caravan,P. et al. Chem. Rev. 1999, 99, 2293). This use is permitted due to thefact that the first coordination sphere of the lanthanide is not fullysaturated with the ligand in aqueous solution, water molecules thusbeing able to complete the coordination sphere.

SUMMARY OF THE INVENTION

The aim of the present invention is to propose lanthanide complexes thathave improved properties over the lanthanide complexes of the prior art.Accordingly, one subject of the invention is novel compounds, their usefor preparing complexes with lanthanide ions, and also the use of thecomplexes obtained as fluorescent markers, and as relaxation agents forNMR or for NMR imaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows droplets about 750 microns in diameter containing BSAmarked with compound 9 (left-hand and right-hand columns on each image)and a fluorescein-marked antibody (middle column on each image) servingas reference (fluorescein-marked rabbit immunoglobulin produced byDako-Immunoglobuline under the product code F-123). The image on theleft was obtained by conventional fluorescence microscopy and the imageon the right was obtained by time-resolved luminescence microscopy.

DETAILED DESCRIPTION

A compound according to the present invention corresponds to formula (I)

in which

-   -   R¹ is a functional group capable of reacting with the functions        present on proteins, antibodies or on mineral or organic        materials;    -   X represents a single bond or a hydrocarbon-based chain        consisting of at least one group chosen from alkylene groups and        alkenylene groups optionally comprising at least one hetero        atom, and from arylene groups;    -   R² is a group A² that is anionic at neutral pH or an alkylene or        alkenylene group containing from 1 to 4 carbon atoms and bearing        at least one such group A², said alkylene or alkenylene group        optionally comprising at least one hetero atom in the chain;    -   R³ represents H or an alkylene or alkenylene group containing        from 1 to 5 carbon atoms and optionally containing at least one        hetero atom in the chain, said group optionally bearing at least        one group A³ that is anionic at neutral pH;    -   R⁴ is a group corresponding to the formula        —(C)_(n)—C—Z¹—C—C—Z²—C—A⁴ in which n is equal to 1 or 2, Z¹ and        Z² represent, independently of each other, a hetero atom chosen        from O and N, at least one being a nitrogen atom forming part of        an aromatic heterocycle with the two carbon atoms surrounding        it, and A⁴ is a group that is anionic at neutral pH, in which        the atom bearing the anionic charge is in the γ position        relative to Z²;    -   R⁵ is a group chosen from the groups defined for R⁴ or from        hydrocarbon-based chains —C—C—E¹—C—C—E²—C—A⁵ in which E¹ and E²        represent, independently of each other, a hetero atom chosen        from O and N, and A⁵ is a group that is anionic at neutral pH,        in which the atom bearing the anionic charge is in the γ        position relative to E².

The hetero atom of the substituents X, R² and R³ may especially be O orN.

The substituent R¹ may be chosen, for example, from amino, thio, cyano,isocyano, acridinyl, hydrazino, haloacetate, anhydride, triazo,carbonyl, nitrobenzoyl, sulfonyl, thionyl, halide, epoxide, aldehyde,imidazole, hydroxyphenyl, mercapto, N-succinimidyl ester,N-sulfosuccinimidyl ester, maleimido, hydroxyl, carboxyl, thiocyano, andisothiocyano groups. The amino, thio, carboxyl, maleimido,N-succinimidyl ester, N-sulfo-succinimidyl ester and isothiocyano groupsare preferred.

When the group X is an alkylene or alkenylene group, it preferablycontains from 1 to 10 carbon atoms. When X is an arylene group, itpreferably contains from 5 to 10 carbon atoms. In the present text, theterm “arylene group” means a group comprising one or more fused orunfused aromatic nuclei, the said nucleus (nuclei) optionally bearingone or more aliphatic hydrocarbon-based groups. Examples of arylenegroups that may be mentioned include the groups —C₆H₄—, —CH₂—C₆H₄—CH₂—,—C₆H₄—CH₂—, —C₆H₄—CH₂—C₆H₄—, —C₆H₃(CH₃)—. X is advantageously chosenfrom a single bond or an alkylene or alkenylene group containing 2 or 3carbon atoms.

The substituent R² is preferably a group A².

The substituent R³ is preferably H or a C₁ to C₃ alkyl.

In the compounds of the present invention, each of the substituents R⁴and R⁵ is a monovalent substituent. The substituents R⁴ and R⁵ do nottogether form a divalent group.

The substituent R⁴ is a substituent that has light-absorbing propertiesand with which three chelate rings can be formed with a lanthanide. Thesubstituents R⁴ in which n is equal to 1 are preferred. As examples ofsubstituents R⁴ in which only one from among Z¹ and Z² is a nitrogenatom that forms part of an aromatic heterocycle, mention may be made ofsubstituents in which one of the segments —C—Z^(i)—C—forms part of aheterocyclic group chosen from pyridyl, pyrimidinyl, quinolyl andisoquinolyl groups. The substituents R⁴ in which Z¹ and Z² form part ofan aromatic heterocyclic group are particularly advantageous. Examplesof such substituents that may be mentioned include substituents in whicheach of the segments —C—Z¹—C— and —C—Z²—C— forms part of a heterocyclicgroup chosen from pyridyl, pyrimidinyl, quinolyl and isoquinolyl groups,the two heterocyclic groups being linked by at least two carbon atomsseparating Z¹ and Z². Examples of such segments —C—Z¹—C—C—Z²—C— that maybe mentioned include 2,2′-bipyridyl, 1,10-phenanthrolinyl,2,2′-bisquinolyl, 2,2′-bisisoquinolyl and 2,2′-bipyrimidinyl groups,said groups possibly bearing alkyl or alkoxy substituents on at leastone carbon atom of a heterocycle, preferably an alkyl or alkoxy groupcontaining from 1 to 5 carbon atoms. By way of example, the formulaebelow represent, respectively, a 2,2′-bipyridyl group bearing acarboxyl, a monoalkylphosphonate, a monoarylphosphonate and aphosphonyl, or a phenanthrolinyl group bearing a carboxyl group.

The substituent R⁵ is a substituent with which three chelate rings canbe formed with a lanthanide. Among the substituents R⁵ consisting of ahydrocarbon-based chain —C—C—E¹—C—C—E²—C—A⁵, mention may be made of thefollowing groups:

in which R⁶ and R⁷ represent alkyl chains preferably containing from 1to 5 carbon atoms and optionally containing one or more hetero atoms.The compounds in which R⁴ and R⁵ are identical are particularlypreferred.

As used herein, the expression “group that is anionic at neutral pH”means a functional group which, at neutral pH, is in anionic form, i.e.bears a negative charge. In a compound of the invention, the groups A²,A³, A⁴ or A⁵ that are anionic at neutral pH may be chosen, independentlyof each other, from —CO₂H, —SO₃H, —P(O)(OR)OH, —P(O)R(OH) and —P(O)(OH)₂groups in which R is an alkyl group (preferably of C₁ to C₃) or an arylgroup (preferably of C₅ to C₉). Depending on the pH of the reactionmedium, the compounds (I) are obtained in cationic, zwitterionic oranionic form. In acidic medium, the nitrogen bearing the substituents R⁴and R⁵, and also optionally the hetero atoms Z¹, Z², E¹ and E², are inprotonated form and the compound is in cationic form. In basic medium,the various groups A^(i) are in the form of salts and the compound is inanionic form. At intermediate pH values, of about 6 to 8, the compoundis in zwitterionic form.

A complex according to the present invention consists of a lanthanideion Ln complexed with a ligand that corresponds to formula (I) above.The lanthanide ion is chosen from europium, terbium, samarium,dysprosium, erbium, ytterbium, neodymium and gadolinium ions. Europium,terbium, samarium or dysprosium will preferably be used if the complexis intended to be used for fluorescence marking, and europium,dysprosium or gadolinium will preferably be used when the complex isintended to be used as a contrast agent for NMRI.

In a complex according to the invention in which R⁴ is—C—C—Z¹—C—C—Z²—C—A⁴, the 3 chelate rings form between the lanthanidecation and, respectively:

-   -   the N atom bearing R⁴ and R⁵, Z¹ and the carbon atoms that        separate them;    -   Z¹, Z² and the two carbon atoms that separate them;    -   the end segment Z²—C—A⁴.

When R⁵ is of the same type as R⁴, it forms with the lanthanide ionchelates of the same type as those formed by R₄. When R⁵ is of the type—C—C—E¹—C—C—E²—C—A⁵, three 5-membered chelate rings form between thelanthanide cation and, respectively:

-   -   the N atom bearing R⁴ and R⁵, E¹ and the two carbon atoms that        separate them;    -   E¹, E² and the two carbon atoms that separate them;    -   the end segment E²—C—A⁵.

A compound (I) may be obtained via processes that are well known tothose skilled in the art from commercial products or products describedin the literature via the following scheme:

in which X, R¹, R², R³, R⁴ and R⁵ have the meaning given above, and R¹′,R²′, R³′, R⁴′ and R⁵′ represent groups that are precursors of R¹, R²,R³, R⁴ and R⁵, respectively.

During the first two steps, the groups R⁴′ and R⁵′ are successivelyintroduced onto a molecule IA containing X and the groups R¹′, R²′ andR³′ to obtain the compound IC.

During subsequent steps, the groups R¹′, R²′, R³′, R⁴′ and R⁵′ of thecompound IC are converted, respectively, into groups R¹, R², R³, R⁴ andR⁵.

When the groups R⁴′ and R⁵′ are identical in order to obtain identicalgroups R⁴ and R⁵, they are introduced simultaneously during the firststep. When they are different, they are introduced in any order byreacting the molecule IA with two different reagents successively.

When compound (I) is a compound in which the groups R¹ and R² arecarboxyl functions, the group R³ is a hydrogen atom and the group X is asingle bond, a methylene group or an ethylene group, the startingmaterial IA that will advantageously be chosen is, respectively, diethylaminomalonate, dimethyl aspartate and dimethyl glutamate, which arecommercially available products.

When compound (I) is a compound in which:

-   -   the groups R¹ and R² are carboxyl functions,    -   the group R³ is a hydrogen atom, and    -   the group X is a propylene or a para-substituted benzene, the        starting material IA that may be used is, respectively, dimethyl        2-aminoadipate (the preparation of which is described by        Lerch, E. et al, Helv. Chim. Acta, 1974, 57, 1584) and methyl        (α-amino-4-methoxycarbonyl)benzene acetate (the preparation of        which is described by Chauvel, E. et al, J. Med. Chem. 1994, 37,        1339).

When the groups R⁴ and R⁵ are identical and their segments—C—C—Z¹—C—C—Z²—C— are derived from 2,2′-bipyridine, the startingmaterial is reacted during the first step with6-bromomethyl-6′-bromo-2,2′-bipyridine to obtain a dibromo compound IC.6-bromomethyl-6′-bromo-2,2′-bipyridine may be obtained via a radicalbromination reaction of 6-methyl-6′-bromo-2,2′-bipyridine withN-bromo-succinimide in benzene, 6-methyl-6′-bromo-2,2′-bipyridine beingobtained according to the method described by Houghton M. et al, J.Chem. Soc., Dalton Trans. 1997, 2725. The reaction scheme for the firststep of this particular case is given below.

When the dibromo compound IC is subjected to a carboalkoxylationfollowed by a saponification with NaOH and an acidification with HCl, acompound (I) is obtained in which the groups A⁴ and A⁵ are carboxylgroups. The carboalkoxylation may be performed according to the processdescribed by El-Ghayoury et al, J. Org. Chem., 2000, 65, 7757.

When the dibromo compound IC is reacted with a dialkyl phosphite(according to the method described by Penicaud et al, Tetrahedron Lett.1998, 39, 3689), the dialkyl-phosphonate is obtained, each bromine atombeing replaced with a group P(O)(OR)₂. The dialkylphosphonate gives, onsaponification with NaOH in water, followed by acidification with HCl, acompound (I) in which the groups A⁴ and A⁵ are groups P(O)(OH)OR.

By reacting the dialkylphosphonate P(O)(OR)₂ with trimethylsilylbromidefollowed by a hydrolysis (according to the method described by McKennaC. et al., Tetrahedron Lett, 1977, 18, 155), a compound (I) is obtainedin which the two anionic groups A⁴ and A⁵ are P(O)(OH)₂ groups. The sameresult may be obtained by means of an acid hydrolysis with HCl of thedialkylphosphonate P(O)(OR)₂.

The reaction scheme for the three operating modes above is given below.

When the groups R⁴ and R⁵ are identical and their segments—C—C—Z¹—C—C—Z²—C— are derived from 1,10-phenanthroline, the startingmaterial is reacted during the first step with2-bromomethyl-9-ethoxycarbonyl-1,10-phenanthroline. The preparation of2-bromomethyl-9-ethoxycarbonyl-1,10-phenanthroline is described byUlrich G. et al, (Tetrahedron Lett. 2001, 42, 6113). By subjecting thediester compound obtained to a saponification with NaOH, followed by anacidification with dilute HCl, a compound (I) is obtained in which thegroups A⁴ and A⁵ are carboxyls. The reaction scheme is given below.

A desired substituent R¹ may be obtained by selecting either a startingcompound that bears it or a starting compound that bears a precursor R¹′of the desired substitutent. When a substituent R¹ is obtained from aprecursor R¹′, the formation of the desired substituent may be performedon a compound of formula (IC) containing the precursor or on a complexformed with a lanthanide cation and a compound of formula (I) containingthe precursor.

A substituent R¹ of the carboxyl type may be obtained via asaponification reaction starting with a precursor group R¹′ containing acarboxylic ester function. A substituent R¹ of the amino type may beobtained from the reduction of a precursor group R¹′ containing a nitrofunction. A substituent R¹ of the isothiocyano type may be obtained byreacting a precursor R¹′ containing an amino function with thiophosgene.A substituent R¹ of the maleimido type may be obtained by reacting aprecursor R¹′ containing an amino function with the N-succinimidyl esterof 4-maleimidobutyric acid.

A substituent R¹ of the N-succinimidyl ester type may be obtained from acomplex by activation of a carboxyl precursor withN-(3-dimethylaminopropyl)-N′-ethylcarbodiimide followed by a reactionwith N-hydroxysuccinimide.

A complex according to the invention may be obtained by reacting acompound giving a lanthanide cation with a compound of formula (I).Examples of compounds giving a lanthanide cation that may be mentionedinclude lanthanide halide hydrates, lanthanide nitrate hydrates,lanthanide carbonates and lanthanide triflates. The reaction isperformed in solution in a solvent. The solvent is preferably chosenfrom water, methanol, ethanol and acetonitrile.

In one preferred embodiment, compound (I) is reacted with the lanthanideion precursor in a mixture of methanol and water at a pH ranging from 3to 5, for a time of between 10 minutes and 24 hours, and at atemperature of between 25° C. and 80° C. Next, the pH of the solution israised to 7.0 and the methanol is evaporated off before isolating thecomplex formed.

The complexes of the present invention may be used especially forfluorescence marking or for nuclear magnetic resonance imaging. Forthese applications, the preferred groups R¹ are amino, thio and carboxylgroups (which must be activated before the covalent coupling with themolecule to be marked), and maleimido, N-succinimidyl ester andisothiocyano groups (which can bind directly with the molecule to bemarked).

The complexes of the present invention are useful for analyses or assaysof compounds by marking of the compounds. The process consists incovalently bonding to the compound to be assayed a marker consisting ofa complex according to the invention, and in detecting or quantifyingthe presence of the marked compound by means of the luminescenceproperties of the marker. Europium, terbium, samarium or dysprosiumcomplexes are particularly preferred for this application.

When the lanthanide ion complexes according to the invention areintended to be used as relaxation agents for nuclear magnetic resonance,gadolinium, europium or dysprosium complexes are preferably used.

The present invention will be described in greater detail by means ofthe examples given below as illustrations, to which it is not, however,limited.

Example 1 Preparation of Compound 1

Compound 1 was obtained according to the synthetic scheme below. The (S)isomer of the chosen glutamic ester may be replaced with the (R) isomeror a mixture of the two isomers.

Preparation of Compound 2

Compound 2 was prepared according to the process described by S. Mameri,et al., in Synthesis, 2003, 17, 2713. 1.5 g (6.0 mmol) of6-methyl-6′-bromo-2,2′-bipyridine, 66 mg (0.4 mmol) ofazobisisobutyronitrile (AIBN) and 1.3 g (7.3 mmol) of N-bromosuccinimideare introduced into 90 mL of benzene in a 250 mL round-bottomed flask.The solution is refluxed for 2 hours 30 minutes by irradiating it with astandard 100 W halogen lamp. The solvent is evaporated off under reducedpressure and the solid residue is chromatographed on silica using aCH₂Cl₂/hexane gradient of from 50/50 to 100/0. 940 mg (2.9 mmol) ofcompound 2 are obtained (corresponding to a yield of 48%), which has thefollowing characteristics:

R_(f)=0.42, SiO₂, CH₂Cl₂.

¹H-NMR (CDCl₃, 200 MHz): δ 4.61 (s, 2H), 7.48 (d, 1H, ³J=7.5 Hz), 7.50(d, 1H, ³J=7.5 Hz), 7.68 (t, 2H, ³J=8.0 Hz), 7.83 (t, 1H, ³J=8.0 Hz),8.33 (d, 1H, ³J=8.0 Hz), 8.44 (d, 1H, ³J=8.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 34.0, 120.1, 120.7, 124.0, 128.2, 138.1,139.3, 141.6, 154.3, 156.4, 156.9.

Analyses calculated for C₁₁H₈N₂Br₂: C, 40.28; H, 2.46; N, 8.54. Found:C, 40.12; H, 2.34; N, 8.44.

FAB⁺/MS: 327 (50%), 329 (100%), 331 (50%, [2+H]⁺).

Preparation of Compound 3

470 mg (2.22 mmol) of dimethyl L-glutamate hydrochloride and 1.23 g ofK₂CO₃ (8.90 mmol) are introduced into 100 mL of acetonitrile freshlydistilled over P₂O₅, in a Schlenk tube under an argon atmosphere. Thesolution is heated at 80° C. for 30 minutes. 1.60 g (4.88 mmol) ofcompound 2 are added and the mixture is heated for 23 hours at 80° C.The solution is evaporated to dryness and the residue is redissolvedwith 100 mL of CH₂Cl₂ and 20 mL of water. The aqueous phase is washedwith two portions of 20 mL of CH₂Cl₂ and the combined organic phases aredried over MgSO₄, filtered, and then evaporated to dryness. The solidresidue is subjected to flash chromatography on silica (φ=5 cm, h=12 cm)with a mixture of CH₂Cl₂/MeOH (100/0 to 97/3) as eluent. 995 mg (1.49mmol) of compound 3 are obtained (corresponding to a yield of 67%),which has the following characteristics:

R_(f)=0.34, SiO₂, CH₂Cl₂/MeOH (98/2).

¹H-NMR (CDCl₃, 200 MHz): δ 2.06-2.20 (m, 2H), 2.39-2.68 (m, 2H), 3.50(s, 3H), 3.54-3.62 (m, 1H), 3.76 (s, 3H), 3.99-4.16 (m, 4H), 7.43-7.48(m, 4H), 7.63 (t, 2H, ³J=8.0 Hz), 7.71 (t, 2H, ³J=8.0 Hz), 8.23 (d, 2H,³J=8.0 Hz), 8.39 (d, 2H, ³J=8.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 24.8, 30.3, 51.5, 57.2, 62.1, 119.6, 119.7,123.5, 127.8, 137.3, 139.1, 141.5, 153.8, 157.4, 159.1, 173.1, 173.4.

Analyses calculated for C₂₉H₂₇N₅O₄Br₂: C, 52.04; H, 4.07; N, 10.46.Found: C, 51.81; H, 3.85; N, 10.19.

FAB⁺/MS: 670.2 ([3+H]⁺, 100%).

Preparation of Compound 4

995 mg of (1.49 mmol) of compound 3 and 150 mg (0.21 mmol) of[Pd(PPh₃)₂Cl₂] are introduced into 50 mL of ethanol and 50 mL oftriethylamine in a 250 mL two-necked round-bottomed flask. The solutionis heated at 70° C. for 15 hours by sparging with a flow of CO. Thesolution is evaporated to dryness, the solid obtained is redissolved in100 mL of CH₂Cl₂ and filtered through Celite, and the organic phase isthen extracted with 20 mL of water. The aqueous phase is washed with twoportions of 20 mL of CH₂Cl₂ and the combined organic phases are driedover MgSO₄, filtered and then evaporated to dryness. The residue issubjected to flash chromatography on silica (φ=5 cm, h=10 cm) with amixture of CH₂Cl₂/MeOH (99/1 to 90/10) as eluent. 588 mg (0.90 mmol) of4 are obtained in the form of a slightly orange-colored oil(corresponding to a yield of 60%), which has the followingcharacteristics:

R_(f)=0.30, SiO₂, CH₂Cl₂/MeOH (95/5).

¹H-NMR (CDCl₃, 200 MHz): δ 1.46 (t, 6H, ³J=7.0 Hz), 2.06-2.19 (m, 2H),2.38-2.65 (m, 2H), 3.49 (s, 3H), 3.55-3.63 (m, 1H), 3.76 (s, 3H),4.02-4.19 (m, 4H), 4.48 (q, 4H, ³J=7.0 Hz), 7.47 (d, 2H, ³J=8.0 Hz),7.75 (t, 2H, ³J=8.0 Hz), 7.92 (t, 2H, ³J=88.0 Hz), 8.10 (d, 2H, ³J=8.0Hz), 8.40 (d, 2H, ³J=8.0 Hz), 8.62 (d, 2H, ³J=8.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 14.3, 24.8, 30.4, 51.5, 57.2, 61.8, 62.0,119.9, 123.5, 124.2, 124.8, 137.3, 137.7, 147.8, 154.6, 156.5, 159.0,165.4, 173.2, 173.5.

Analyses calculated for C₃₅H₃₇N₅O₈: C, 64.11; H, 5.69; N, 10.68. Found:C, 64.07; H, 5.55; N, 10.53.

FAB⁺/MS: 656.2 ([4+H]⁺, 100%).

Preparation of Compound 1

588 mg (0.90 mmol) of 4 and 144 mg (3.60 mmol) of NaOH are dissolved ina mixture of 50 mL of MeOH and 15 mL of water in a round-bottomed flaskequipped with a condenser. The mixture is heated at 70° C. for 5 hours.The solution is evaporated to dryness and the solid is dissolved in 10mL of water to which is slowly added 2N HCl solution to pH 2-3. Theprecipitate formed is isolated by centrifugation and dried under vacuum.411 mg (0.60 mmol) of compound 1 are obtained in the form of a paleyellow hydrochloride 1.3 HCl (corresponding to a yield of 67%), thecharacteristics of which are as follows:

¹H-NMR (CD₃OD, 300 MHz): δ 2.26-2.48 (m, 2H), 2.80-2.84 (m, 2H),3.95-3.99 (m, 1H), 4.53-4.81 (m, 4H), 7.47 (d, 2H, ³J=7.5 Hz), 7.63 (t,2H, ³J=8.0 Hz), 7.90 (t, 2H, ³J=8.0 Hz), 8.02 (d, 2H, ³J=7.5 Hz), 8.42(d, 2H, ³J=7.5 Hz), 8.58 (d, 2H, ³J=7.5 Hz).

¹³C-NMR (CD₃OD, 75 MHz): δ 23.1, 32.1, 57.0, 67.0, 122.3, 125.1, 125.9,126.1, 139.7, 140.1, 149.0, 154.1, 155.5, 156.1, 168.0, 173.7, 176.4.

Analyses calculated for C₂₉H₂₅N₅O₈.3HCl: C, 51.15; H, 4.14; N, 10.28.Found: C, 51.01; H, 4.43; N, 9.95.

FAB⁺/MS: 572.5 ([1+H]⁺, 100%).

Example 2 Preparation of Complex 5 of Formula [Eu.(1-4H).H₂O]Na

60 mg of 1.3 HCl (88 μmol) are dissolved in a mixture of 30 mL of MeOHand 30 mL of water. To this solution is added a mixture of 36 mg (98μmol) of EuCl₃.6H₂O dissolved in 3 mL of MeOH and 3 mL of water. Thesolution is heated at 70° C. for 1 hour. After cooling, the pH of thesolution is raised to 7.4 with a 5% solution of NaOH in water. Thesolution is concentrated on a rotary evaporator until slight cloudinessappears. THF is then added until a substantial precipitate forms. Theprecipitate is isolated by centrifugation and then dried under vacuum togive 62 mg (74 μmol) of compound 5 (corresponding to a yield of 85%) inthe form of a beige-colored solid, the characteristics of which are asfollows:

¹H-NMR (D₂O/t-BuOH, 200 MHz, all the signals are in the form of broadsinglets): δ −9.40 (1H), −8.95 (1H), −4.23 (2H), −3.17 (1H), −2.21 (1H),1.88 (1H), 2.73 (1H), 4.17 (1H), 6.06 (1H), 7.12 (1H), 7.80 (1H), 7.88(1H), 8.90 (1H), 9.60 (1H), 9.89 (1H), 11.08 (1H), 11.38 (1H), 12.01(1H).

Analyses calculated for C₂₉H₂₁NaN₅O₈Eu.5H₂O: C, 41.84; H, 3.75; N, 8.41.Found: C, 41.93; H, 3.62; N, 8.44.

FAB⁺/MS: 720.2 (80%), 722.2 (100%), [5-H₂O—Na+2H]⁺.

IR (KBr, cm⁻¹): 3420, 1619, 1574, 1460, 1384, 1274.

Photophysical properties in water:

Absorption, λ_(max) [nm] (ε_(max) [M⁻¹.cm⁻¹]): 320 (shoulder), 308 (19700), 276 (8700), 267 (9700), 253 (14 400).

Emission: characteristic of europium compounds with fine bands at 581,594, 615, 650 and 701 nm. Lifetime of the excited state: 0.62 ms.Quantum yield (reference [Ru(bipy)₃]²⁺ in water): 8%. Lifetime of theexcited state in deuterated water: 2.48 ms. Quantum yield in deuteratedwater: 35%.

Example 3 Preparation of Complex 6 of Formula

40 mg (48 μmol) of complex 5 and 12 mg (63 μmol) ofethyl-N,N-dimethyl-3-aminopropylcarbodiimide hydrochloride (EDCI.HCl)are suspended in 6 mL of DMSO. To this solution are added 7.0 mg (61μmol) of N-hydroxysuccinimide. The solution is stirred at roomtemperature for 66 hours, during which the complex 5 dissolves, and awhite precipitate then forms. The solid is isolated by centrifugationand dried under vacuum at 50° C. for 2 hours. 31 mg (34 μmol) of 6 areobtained (corresponding to a yield of 71%), the characteristics of whichare as follows:

Analyses calculated for C₃₃H₂₅EuN₆O₁₀.5H₂O: C, 43.67; H, 3.89; N, 9.26.Found: C, 43.60; H, 3.80; N, 9.16.

FAB⁺/MS: 720.1, 722.1 ([6-H₂O—C₄H₄NO₂+2H]⁺, 100%), 817.1, 819.1([6-H₂O+H]⁺, 30%).

IR (KBr disk, cm⁻¹): 3420, 1739, 1629, 1573, 1459, 1384.

Photophysical properties in water:

Absorption, λmax [nm] (ε_(max) [M⁻¹.cm⁻¹]): 320 (shoulder), 309 (20000), 276 (10 000), 267 (10 500), 253 (16 000).

Emission: characteristic of europium compounds with fine bands at 581,593, 615, 649 and 701 nm. Lifetime of the excited state: 0.63 ms.Quantum yield (reference [Ru(bipy)₃]²⁺ in water): 8%. Lifetime of theexcited state in deuterated water: 2.47 ms. Quantum yield in deuteratedwater: 34%.

Example 4 Marking of an Amine with Complex 5

10 mg of complex 5 (13.1 μmol) are suspended in 5 mL of water. 3.5 mg(18.3 μmol) of EDCI.HCl and then 1.7 μL (13.2 μmol) of(+)-α-methylbenzylamine are added. After 15 minutes, and then after onehour, 1.7 μL of (+)-α-methyl-benzylamine are added each time, at roomtemperature. Stirring is continued for 15 hours. The aqueous phase iswashed with twice 10 mL of CH₂Cl₂ and then evaporated to dryness to give14 mg of a pale yellow solid. After recrystallization from an MeOH/Et₂Omixture, centrifugation and drying under vacuum, complex 7 (8.0 mg, 9.5μmol) is recovered in the form of a cream-colored powder (73%).

ISI-TOF/MS: 847.0513 ([7-H₂O+Na]⁺, 60%), 825.0912 ([7-H₂O+H]⁺, 28%). Theformula of complex 7 is shown below.

Example 5 Marking of Bovine Serum Albumin BSA with Complex 6

Complex 6 (2.0 mg) is added to a solution of BSA (5.4 mg) in 1 mL ofborate buffer (50 mM in water, pH=7.0) in order to obtain a 6/ASB moleratio of 30:1. The solution is stirred at room temperature, leading tototal dissolution of 6 after 2 hours. After stirring for 24 hours, thesolution is deposited on a centrifuge filter (Centricon, Millipore, 30KDa filter) and the volume of the solution is reduced to 200-300 μL byfiltration. The solution is diluted with 3 mL of water and the volume isagain reduced to 200-300 μL by filtration. This last operation isrepeated 3 to 4 times, until the filtration waters are no longerluminescent under UV irradiation (absence of europium). The 200-300 μLof residual solution containing the marked protein and remaining on thefilter are recovered and stored in a refrigerator at 4° C.

Characterization of the Marked BSA

The UV-Vis absorption spectrum of the aqueous solution of marked BSAshows a strong absorption due to the europium complexes, which partiallyoverlaps the absorption due to the protein (λ_(max)=278 nm, ε_(max)=38000 M⁻¹.cm⁻¹). On excitation of the solution in the absorption band ofthe bipyridines (308 nm), a typical emission spectrum of europiumcompounds is observed, with a mean lifetime of the excited state of 1.1ms (the decrease is not purely mono-exponential) and a luminescencequantum yield of 13% is observed.

Characterization by mass spectrometry in MALDI-TOF mode (MatriceAssisted Laser Desorption Ionization-Time Of Fly) is performed in thefollowing manner. An aqueous solution of marked BSA is treated with 1%trifluoroacetic acid to decomplex the europium, and the protein is thenadsorbed onto a chromatography column whose hydrophobic solid phaseconsists of C₄ chain. After washing with water, the protein is releasedwith acetonitrile and then analyzed by MALDI-TOF(α-cyano-4-hydroxycinnamic acid matrix). The mean mass obtained for theeuropium-free marked protein is 71 700 Da (BSA, M=66 610 Da), leading toa markers/BSA mole ratio of 9/1 in the marked protein.

Example 6 Preparation of Complex 8 of Formula [Tb.(1-4H).H₂O]Na

40 mg (59 μmol) of compound 1.3 HCl are dissolved in a mixture of 30 mLof MeOH and 30 mL of water in a 250 mL round-bottomed flask equippedwith a condenser. To this solution are added 25 mg (67 μmol) ofTbCl₃.6H₂O dissolved in 5 mL of MeOH and 5 mL of water. The solution isheated at 70° C. for one hour. After cooling, the pH of the solution israised to 7.2 with a 1% solution of NaOH in water. The solution isconcentrated on a rotary evaporator until slight cloudiness appears, andTHF is then added until a substantial precipitate forms. A pale yellowsolid is isolated by centrifugation and then dried under vacuum. 46 mg(56 μmol) of complex 8 are obtained (corresponding to a yield of 95%),the characteristics of which are as follows:

Analyses calculated for C₂₉H₂₁NaN₅O₈Tb.4H₂O: C, 42.40; H, 3.56; N, 8.53.Found: C, 42.28; H, 3.31; N, 8.38.

FAB⁻/MS: 668.2 ([8-H₂O—CH₂COONa]⁻, 100%), 726.2 ([8-H₂O—Na]⁻, 30%).

IR (KBr disk, cm⁻¹): 3428, 1592, 1574, 1466, 1416, 1387.

Photophysical properties in water:

Absorption, λ_(max) [nm] (ε_(max) [M⁻¹.cm⁻¹]): 320 (shoulder), 308 (20800), 277 (8900), 267 (10 400), 253 (15 000).

Emission: characteristic of terbium compounds with fine bands at 487,543, 583 and 621 nm. Lifetime of the excited state: 1.48 ms. Quantumyield (reference: quinine sulfate in 1N H₂SO₄): 31%. Lifetime of theexcited state in deuterated water: 2.53 ms. Quantum yield in deuteratedwater: 53%.

Example 7 Preparation of Complex 9 of Formula

50 mg (61 μmol) of complex 8 are suspended in 5 mL of DMSO in a 10 mLround-bottomed flask. To this solution are added 9 mg (78 μmol) ofN-hydroxysuccinimide and 13 mg (68 μmol) ofethyl-N,N-dimethyl-3-aminopropylcarbodiimide hydrochloride (EDCI.HCl).The solution is stirred at room temperature for 138 hours, during whichtime complex 8 dissolves and a white precipitate then forms. The solidis isolated by centrifugation, washed with THF and dried under vacuum.Addition of THF to the mother liquors causes the formation of a furtherprecipitate, which is recovered by centrifugation. 49 mg (55 μmol) ofcomplex 9 are obtained in total (corresponding to a yield of 90%), thecharacteristics of which are as follows:

Analyses calculated for C₃₃H₂₅N₆O₁₀Tb.4H₂O: C, 44.21; H, 3.71; N, 9.29.Found: C, 44.01; H, 3.42; N, 9.29.

FAB⁺/MS: 726.2 ([9-H₂O—C₄H₄NO₂]⁺, 15%), 825.5 ([9-H₂O+H]⁺, 100%).

IR (KBr disk, cm⁻¹): 3433, 1741, 1624, 1594, 1574, 1464, 1419, 1375.

Photophysical Properties in Water

Absorption, λ_(max) [nm] (ε_(max) [M⁻¹.cm⁻¹]): 308 (18 700), 276, 267,253.

Emission: characteristic of terbium compounds with fine bands at 487,543, 583 and 621 nm. Lifetime of the exited state: 1.50 ms. Quantumyield (reference: quinine sulfate in 1N H₂SO₄): 34%. Lifetime of theexcited state in deuterated water: 2.42 ms. Quantum yield in deuteratedwater: 62%.

Example 8 Marking of Bovine Serum Albumin BSA with Complex 9 andRevelation by Time-Resolved Luminescence Microscopy

The marking of the bovine serum albumin was performed according to themethod described in Example 5, replacing the complex 6 with complex 9.

Determination of the Markers/BSA Mole Ratio

The markers/BSA mole ratio (number of complexes 9 covalently bonded toBSA) is determined by differential absorption at 308 nm. The molarabsorption coefficients of native BSA and of the marked BSA are measuredat 308 nm. The difference between these two values is divided by themolar absorption coefficient of 9 at 308 nm, to give a markers/BSA moleratio of 6/1 in the marked protein.

FIG. 1 shows droplets about 750 microns in diameter containing BSAmarked with compound 9 (left-hand and right-hand columns on each image)and a fluorescein-marked antibody (middle column on each image) servingas reference (fluorescein-marked rabbit immunoglobulin produced byDako-Immunoglobuline under the product code F-123). The image obtainedby conventional fluorescence microscopy (left) reveals the fluorescenceof the two compounds. The image obtained by time-resolved luminescencemicroscopy (delay=0.5 ms, integration time=5.0 ms) shows thedisappearance of the fluorescence of the reference compound, whereas theluminescence of the marked BSA persists.

Example 9 Preparation of Complex 10 of Formula [Gd.(1-4H).H₂O]Na

30 mg (44 μmol) of compound 1.3 HCl are dissolved in a mixture of 25 mLof MeOH and 25 mL of water in a 100 mL round-bottomed flask equippedwith a condenser. To this solution are added 19 mg (51 μmol) ofGdCl₃.6H₂O dissolved in 5 mL of MeOH and 5 mL of water. The solution isheated at 70° C. for one hour. After cooling, the pH of the solution israised to 7.5 with a 0.5% solution of NaOH in water. The solution isconcentrated on a rotary evaporator until slight cloudiness appears, andTHF is then added until a substantial precipitate forms. The pale yellowsolid is isolated by centrifugation and then dried under vacuum to give30 mg (37 μmol) of complex 10 (corresponding to a yield of 85%), thecharacteristics of which are as follows:

Analyses calculated for C₂₉H₂₁GdNaN₅O₈.3H₂O: C, 43.44; H, 3.39; N, 8.73.Found: C, 43.35; H, 3.17; N, 8.55.

FAB⁻/MS: 667.2 ([10-H₂O—CH₂COONa]⁻, 100%), 725.2 ([10-H₂O—Na]⁻, 45%).

IR (KBr disk, cm⁻¹): 3422, 1637, 1592, 1459, 1419, 1385.

Example 10 Preparation of Complex 11 of Formula

50 mg (62 μmol) of compound 10 are suspended in 5 mL of DMSO in a 10 mLround-bottomed flask. To this solution are added 9 mg (78 μmol) ofN-hydroxysuccinimide and 15 mg (78 μmol) ofethyl-N,N-dimethyl-3-aminopropylcarbodiimide hydrochloride (EDCI.HCl).The solution is stirred at room temperature for 48 hours, during whichtime complex 10 dissolves and a white precipitate then forms. The solidis isolated by centrifugation, washed with THF and dried under vacuum.The addition of THF to the mother liquors causes the formation ofadditional precipitate, which is recovered by centrifugation. 45 mg (51μmol) of complex 11 are obtained in total (corresponding to a yield of82%), the characteristics of which are as follows:

Analyses calculated for C₃₃H₂₅GdN₆O₁₀.3H₂O: C, 45.20; H, 3.56; N, 9.37.Found: C, 45.02; H, 3.18; N, 9.21.

FAB⁺/MS: 726.5 ([11-H₂O—C₄H₄NO₂+2H]⁺, 20%), 824.2 ([11-H₂O+H]⁺, 100%).

IR (KBr disk, cm⁻¹): 3435, 1741, 1623, 1573, 1465, 1420, 1376.

Example 11 Preparation of Compound 12

This compound is obtained in two steps from compound 3 according to thefollowing synthetic scheme:

Preparation of Compound 13

200 mg (0.30 mmol) of compound 3, 90 μL (0.70 mmol) of diethylphosphite, 78 mg (0.30 mmol) of PPh₃ and 300 μL of freshly distilleddiisopropylethylamine are introduced into 10 mL of toluene in a Schlenktube under an argon atmosphere. The solution is degassed with argon for20 minutes. 34 mg (0.03 mmol) of Pd(PPh₃)₄ are added and the solution isheated at 100° C. for 16 hours. 40 μL (0.31 mmol) of diethyl phosphiteand 34 mg (0.03 mmol) of Pd(PPh₃)₄ are added and the solution is againheated at 100° C. for 16 hours. The solution is evaporated to dryness.The solid residue is purified by flash chromatography on silica (φ=3 cm,h=15 cm) with a CH₂Cl₂/MeOH mixture (99/1 to 95/5) as eluent. The purefractions are evaporated, dissolved in 30 mL of CH₂Cl₂ and washed with10 mL of water. The organic phase is dried over MgSO₄, filtered andevaporated. 72 mg (0.09 mmol) of compound 13 are obtained (correspondingto a yield of 31%) in the form of an oil having the followingcharacteristics:

R_(f)=0.56, SiO₂, CH₂Cl₂/MeOH (90/10).

¹H-NMR (CDCl₃, 200 MHz): δ 1.35 (t, 12H, ³J=7.0 Hz), 2.02-2.22 (m, 2H),2.37-2.71 (m, 2H), 3.47 (s, 3H), 3.54-3.61 (m, 1H), 3.75 (s, 3H),4.01-4.17 (m, 4H), 4.18-4.36 (m, 8H), 7.47 (d, 2H, ³J=7.5 Hz), 7.73 (t,2H, ³J=8.0 Hz), 7.81-7.97 (m, 4H), 8.32 (d, 2H, ³J=7.5 Hz), 8.59 (dt,2H, ³J_(H-H)=7.0 Hz, ³J_(H-P)=4J_(H-H)=2.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 16.3, 16.4, 24.7, 30.3, 51.4, 57.1, 61.9,63.0, 63.1, 119.6, 123.2 (2), 123.4, 127.4, 127.9, 136.7, 137.0, 137.2,149.0, 153.5, 154.5, 156.5, 156.9, 159.0, 173.1, 173.4.

³¹P-NMR (CDCl₃, 162 MHz): δ 11.73.

Preparation of Compound 12

51 mg (65 μmol) of compound 13 are dissolved in 6 mL of a 0.05 Nsolution of NaOH in water, in a 50 mL round-bottomed flask equipped witha condenser. The mixture is heated at 100° C. for 19 hours. Aftercooling, the aqueous phase is extracted with 4 portions of 5 mL ofCH₂Cl₂ and then evaporated to dryness. The product precipitates from anH₂O/THF mixture. 45 mg (51 μmol) of compound 12 are obtained(corresponding to a yield of 79%) in the form of a cream-colored powder,the characteristics of which are as follows:

¹H-NMR (D₂O/^(t)BuOH, 300 MHz): δ 1.18 (t, 6H, ³J=7.0 Hz), 2.06-2.27 (m,2H), 2.37-2.58 (m, 2H), 3.50 (t, 3H, ³J=7.5 Hz), 3.86-3.99 (m, 4H),4.02-4.24 (m, 4H), 7.48 (d, 2H, ³J=7.0 Hz), 7.59-7.81 (m, 10H).

¹³C-NMR (D₂O/^(t)BuOH, 75 MHz): δ 16.4, 16.5, 27.8, 35.6, 59.8, 62.4,62.5, 71.6, 121.2, 124.0, 124.1, 125.7, 127.1, 127.4, 138.0, 138.2,138.5, 154.6, 155.0, 156.3, 156.6, 157.8, 160.6, 181.1, 183.6.

³¹P-NMR (D₂O, 162 MHz): δ 10.17.

Analyses calculated for C₃₁H₃₁N₅Na₄O₁₀P₂.5H₂O: C, 42.43; H, 4.71; N,7.98. Found: C, 42.35; H, 4.55; N, 7.78.

FAB⁺/MS: 764.2 ([12-Na]⁺, 10%).

Example 12 Preparation of Complex 14 of Formula

19 mg (22 μmol) of compound 12 are dissolved in 35 mL of water in a 50mL round-bottomed flask equipped with a condenser. The pH is adjusted to3.1 with dilute HCl solution. To this solution are added 9 mg (25 μmol)of EuCl₃.6H₂O dissolved in 5 mL of water. The solution is heated at 80°C. for one hour. After cooling, the solution is filtered through Celiteand the pH is raised to 7.1 with a 0.5% solution of NaOH in water. Thesolution is evaporated to dryness and the product precipitates from anH₂O/THF mixture. The pale yellow solid is isolated by centrifugation andthen dried under vacuum, to give 9 mg (10 μmol) of complex 14(corresponding to a yield of 47%), the characteristics of which are asfollows:

FAB⁺/MS: 848.2 ([14-H₂O—Na]⁺, 35%).

Example 13 Preparation of Compound 15

This compound is obtained in three steps according to the followingsynthetic scheme:

Preparation of Compound 16

450 mg (2.13 mmol) of diethyl aminomalonate hydrochloride and 1.18 g(8.54 mmol) of K₂CO₃ are introduced into 150 mL of freshly distilledacetonitrile in a 500 mL round-bottomed Schlenk flask under an argonatmosphere. The solution is heated at 80° C. for one hour. 1.46 g (4.45mmol) of compound 2 are added and the mixture is heated for 21 hours at80° C. The solution is evaporated to dryness and the residue isredissolved with 100 mL of CH₂Cl₂ and 20 mL of water. The aqueous phaseis washed with two portions of 20 mL of CH₂Cl₂ and the combined organicphases are dried over MgSO₄, filtered and then evaporated to dryness.The solid residue is purified by flash chromatography on silica (φ=4 cm,h=14 cm) with a CH₂Cl₂/MeOH mixture (100/0 to 99/1) as eluent. 794 mg(1.19 mmol) of compound 16 are obtained (corresponding to a yield 56%)in the form of a pale yellow powder having the followingcharacteristics:

R_(f)=0.57, SiO₂, CH₂Cl₂/MeOH (97/3).

¹H-NMR (CDCl₃, 200 MHz): δ 1.26 (t, 6H, ³J=7.0 Hz), 4.22 (s, 4H), 4.23(q, 4H, ³J=7.0 Hz), 4.47 (s, 1H), 7.43 (dd, 2H, ³J=7.5 Hz, ⁴J=0.5 Hz),7.60 (t, 2H, ³J=7.5 Hz), 7.62 (d, 2H, ³J=7.5 Hz), 7.75 (t, 2H, ³J=8.0Hz), 8.22 (dd, 2H, ³J=7.5 Hz, ⁴J=1.0 Hz), 8.37 (dd, 2H, ³J=7.5 Hz,⁴J=1.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 14.1, 58.0, 61.4, 67.1, 119.7, 123.4, 127.7,137.4, 139.0, 141.4, 153.5, 157.4, 158.9, 168.1.

Analyses calculated for C₂₉H₂₇Br₂N₅O₄: C, 52.04; H, 4.07; N, 10.46.Found: C, 51.93; H, 3.93; N, 10.31.

FAB⁺/MS: 670.2 (100%), 672.2 (50%), [16+H]⁺.

Preparation of Compound 17

778 mg (1.16 mmol) of compound 16 and 82 mg (0.12 mmol) of[Pd(PPh₃)₂Cl₂] are introduced into 75 mL of ethanol and 75 mL oftriethylamine in a 250 mL two-necked round-bottomed flask. The solutionis heated at 70° C. for 16 hours while sparging with a stream of CO. Thesolution is evaporated to dryness, the solid obtained is redissolved in75 mL of CH₂Cl₂ and filtered through Celite, and the organic phase isthen washed with 15 mL of water. The aqueous phase is extracted with twoportions of 20 mL of CH₂Cl₂ and the combined organic phases are driedover MgSO₄, filtered and then evaporated to dryness. The residue ispurified by flash chromatography on silica ((φ=3 cm, h=16 cm) with aCH₂Cl₂/MeOH mixture (99.5/0.5 to 90/10) as eluent. The fractionscontaining compound 17 with triphenylphosphine oxide are dissolved in 40mL of CH₂Cl₂ and extracted with four portions of HCl 3N. The combinedaqueous phases are neutralized with NaOH and then extracted with threeportions of 30 mL of CH₂Cl₂. The combined organic phases are dried overMgSO₄, filtered and then evaporated to dryness. 522 mg (0.80 mmol) ofcompound 17 are obtained in the form of a colorless oil (correspondingto a yield of 68%), which has the following characteristics:

R_(f)=0.55, SiO₂, CH₂Cl₂/MeOH (90/10).

¹H-NMR (CDCl₃, 200 MHz): δ 1.26 (t, 6H, ³J=7.0 Hz), 1.45 (t, 6H, ³J=7.0Hz), 4.23 (q, 4H, ³J=7.0 Hz), 4.24 (s, 4H), 4.47 (q, 4H, ³J=7.0 Hz),4.48 (s, 1H), 7.64 (dd, 2H, ³J=7.5 Hz, ⁴J=0.5 Hz), 7.80 (t, 2H, ³J=8.0Hz), 7.91 (t, 2H, ³J=7.5 Hz), 8.09 (dd, 2H, ³J=7.5 Hz, ⁴J=1.0 Hz), 8.40(dd, 2H, ³J=7.5 Hz, ⁴J=0.5 Hz), 8.62 (dd, 2H, ³J=8.0 Hz, ⁴J=1.5 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 14.1, 14.3, 58.0, 61.4, 61.8, 67.1, 120.0,123.4, 124.2, 124.7, 137.5, 137.7, 147.7, 154.4, 156.5, 158.8, 165.3,168.2.

Analyses calculated for C₃₅H₃₇N₅O₈: C, 64.11; H, 5.69; N, 10.68. Found:C, 63.81; H, 5.43; N, 10.43.

FAB⁺/MS: 496.2 (35%), 656.1 ([17+H]⁺, 100%).

Preparation of Compound 15

103 mg (0.16 mmol) of compound 17 and 50 mg (1.25 mmol) of NaOH aredissolved in a mixture of 10 mL of MeOH and 5 mL of water in a 50 mLround-bottomed flask equipped with a condenser. The mixture is heated at70° C. for 5 hours. The solution is evaporated to dryness and the solidis dissolved in 8 mL of water, to which is slowly added, at 0° C., 1NHCl solution until an abundant precipitate of the product is obtained(pH=4-5). The precipitate is isolated by centrifugation and dried undervacuum. 59 mg (0.08 mmol) of 15.3HCl hydrochloride hydrate are obtained(corresponding to a yield of 53%) in the form of a white powder, thecharacteristics of which are as follows:

¹H-NMR (NaOD/^(t)BuOH, 300 MHz: δ 3.75 (s, 4H), 4.04 (s, 1H), 6.84 (d,2H, ³J=7.5 Hz), 7.15-7.26 (m, 4H), 7.32 (d, 2H, ³J=7.5 Hz), 7.42 (t, 2H,³J=7.5 Hz), 7.56 (d, 2H, ³J=7.5 Hz). ¹³C-NMR (NaOD/^(t)BuOH, 75 MHz): δ60.3, 79.4, 119.9, 122.9, 124.1, 124.4, 138.2, 138.6, 152.8, 153.7,154.0, 158.7, 168.6, 172.3, 177.3.

Analyses calculated for C₂₇H₂₁N₅O₈.3HCl.3H₂O: C, 45.87; H, 4.28; N,9.91. Found: C, 45.75; H, 4.09; N, 9.78.

FAB⁺/MS: 544.2 ([15+H]⁺, 20%).

Example 14 Preparation of Complex 18 of Formula

15 mg of 15.3HCl.3H₂O (21 μmol) are dispersed in a mixture of 10 mL ofMeOH and 10 mL of water in a 10 mL round-bottomed flask. To thissolution are added 10 mg (27 μmol) of EuCl₃.6H₂O dissolved in 5 mL ofMeOH and 5 mL of water. The solution is heated at 70° C. for one hour.After cooling, the pH of the solution is raised to 7.3 with a 0.5%solution of NaOH in water. The solution is concentrated on a rotaryevaporator until a precipitate forms. The white solid is isolated bycentrifugation and then dried under vacuum to give 14 mg (19 μmol) ofcompound 18 (corresponding to a yield of 90%), the characteristics ofwhich are as follows: FAB⁻/MS: 692.3 ([18-H₂O—Na)]⁻, 100%).

IR (KBr disk, cm⁻¹): 3442, 1626, 1588, 1460, 1411, 1373.

Example 15 Preparation of Complex 19 of Formula

18 mg (22 μmol) of complex 5 are suspended in 5 mL of DMSO in a 10 mLround-bottomed flask. To this solution are added 6 mg (26 μmol) of themonosodium salt of N-hydroxy-succinimide-3-sulfonic acid hydrate and 5mg (26 mol) of ethyl-N,N-dimethyl-3-aminopropylcarbodiimidehydrochloride (EDCI.HCl). The solution is stirred at room temperaturefor 46 hours, during which time complex 5 dissolves. The addition of THFto the solution causes the formation of a precipitate, which isrecovered by centrifugation. 15 mg (15 μmol) of complex 19 are obtained(corresponding to a yield of 68%) in the form of a white powder.

Example 16 Preparation of Complex 20 of Formula

45 mg (55 μmol) of complex 8 are suspended in 10 mL of DMSO in a 50 mLround-bottomed flask. To this solution are added 14 mg (60 μmol) of themonosodium salt of N-hydroxy-succinimide-3-sulfonic acid hydrate and 12mg (63 μmol) of ethyl-N,N-dimethyl-3-aminopropylcarbodiimidehydrochloride (EDCI.HCl). The solution is stirred at room temperaturefor 92 hours, during which time complex 8 dissolves. The addition of THFto the solution causes the formation of a precipitate, which isrecovered by centrifugation. 45 mg (44 μmol) of complex 20 are obtained(corresponding to a yield of 81%) in the form of a yellow powder, thecharacteristics of which are as follows:

Analyses calculated for C₃₃H₂₄N₆NaO₁₃STb.5H₂O: C, 38.99; H, 3.37; N,8.27. Found: C, 39.20; H, 3.56; N, 8.39.

FAB⁺/MS: 682.2 ([20-H₂O—C₅H₃NNaO₇S]⁺, 95%), 727.2([20-H₂O—C₄H₃NNaO₅S+H]⁺, 55%).

Example 17 Preparation of Complex 21 of Formula

19 mg (24 μmol) of complex 10 are suspended in 5 mL of DMSO in a 10 mLround-bottomed flask. To this solution are added 7 mg (30 μmol) of themonosodium salt of N-hydroxy-succinimide-3-sulfonic acid hydrate and 5mg (26 μmol) of ethyl-N,N-dimethyl-3-aminopropylcarbodiimidehydrochloride (EDCI.HCl). The solution is stirred at room temperaturefor 24 hours, during which time complex 10 dissolves. The addition ofTHF to the solution causes the formation of a precipitate, which isrecovered by centrifugation. 19 mg (19 μmol) of complex 21 are obtained(corresponding to a yield of 80%) in the form of a yellow powder, thecharacteristics of which are as follows:

FAB⁺/MS: 681.2 ([21-H₂O—C₅H₃NNaO₇S]⁺, 100%), 726.3([21-H₂O—C₄H₃NNaO₅S+H]⁺, 40%).

Example 18 Preparation of Compound 25

Compound 25 is obtained in four steps according to the following scheme:

Preparation of Compound 22

2.04 g (7.6 mmol) of 7-hydroxy-9-carbomethoxy-2-methylphenanthroline(obtained according to Heindel, N. et al, J. Heterocycl. Chem. 1968, 5,869), 2.11 g (15.2 mmol) of K₂CO₃ and 950 μL (15.3 mmol) of methyliodide are introduced into 60 ml of acetonitrile freshly distilled overP₂O₅, in a Schlenk tube under an argon atmosphere. The solution isheated at 80° C. for 19 hours. The solution is evaporated to dryness andthe residue is dissolved in 100 mL of CH₂Cl₂ and 15 mL of water. Theaqueous phase is extracted with 4 portions of 15 mL of CH₂Cl₂ and thecombined organic phases are dried over MgSO₄, filtered and thenevaporated to dryness. The residue is purified by chromatography onalumina (φ=5 cm, h=12 cm) with a CH₂Cl₂/MeOH mixture (99/1) as eluent.2.05 g (7.3 mmol) of compound 22 are obtained (corresponding to a yieldof 95%) in the form of a yellow powder having the followingcharacteristics:

R_(f)=0.54, Al₂O₃, CH₂Cl₂/MeOH (98/2).

¹H-NMR (CDCl₃, 200 MHz): δ 2.91 (s, 3H), 4.06 (s, 3H), 4.12 (s, 3H),7.47 (d, 1H, ³J=8.5 Hz), 7.77 (d, 1H, ³J=9.0 Hz), 7.83 (s, 1H), 8.08 (d,1H, ³J=7.5 Hz), 8.12 (d, 1H, ³J=9.0 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 25.8, 52.8, 56.2, 102.9, 118.7, 122.2, 123.9,126.9, 127.4, 136.0, 145.2, 146.1, 148.6, 160.1, 163.2, 166.5.

Analyses calculated for C₁₆H₁₄N₂O₃: C, 68.07; H, 5.00; N, 9.92. Found:C, 67.92; H, 4.93; N, 9.78.

FAB⁺/MS: 283.2 ([22+H]⁺, 100%).

Preparation of Compound 23

1 g (3.5 mmol) of compound 22, 630 mg (3.5 mmol) of N-bromosuccinimideand 30 mg (0.2 mmol) of azobis-isobutyronitrile (AIBN) are introducedinto 10 mL of benzene in a 250 mL round-bottomed flask. The solution isirradiated for 30 minutes with a standard 100 W halogen lamp. Thesolvent is evaporated off under reduced pressure and the residue ispurified by chromatography on alumina containing 10% water with amixture of CH₂Cl₂/hexane (50/50) as eluent. 468 mg (1.3 mmol) ofcompound 23 are obtained (corresponding to a yield of 37%) in the formof a gray powder having the following characteristics:

R_(f)=0.71, Al₂O₃, CH₂Cl₂/MeOH (98/2).

¹H-NMR (CDCl₃, 200 MHz): δ 4.06 (s, 3H), 4.12 (s, 3H), 4.93 (s, 2H),7.77 (d, 1H, ³J=9.0 Hz), 7.83 (s, 1H), 7.87 (d, 1H, ³J=8.5 Hz), 8.17 (d,1H, ³J=9.0 Hz), 8.21 (d, 1H, ³J=8.5 Hz).

¹³C-NMR (CDCl₃, 50 MHz): δ 34.6, 53.0, 56.3, 103.3, 120.2, 122.4, 123.7,127.0, 128.1, 137.1, 144.5, 145.9, 148.9, 157.6, 163.3, 166.2.

Analyses calculated for C₁₆H₁₃BrN₂O₃: C, 53.21; H, 3.63; N, 7.76. Found:C, 52.94; H, 3.26; N, 7.51.

FAB⁺/MS: 281.2 ([23-Br]⁺, 30%), 361.2 (100%), 363.2 (100%), [23+H]⁺.

Preparation of Compound 24

96 mg (0.45 mmol) of dimethyl DL-glutamate hydrochloride and 250 mg(1.81 mmol) of K₂CO₃ are introduced into 15 mL of acetonitrile freshlydistilled over P₂O₅, in a Schlenk tube under an argon atmosphere. Thesolution is heated at 80° C. for 10 minutes. 360 g (1 mmol) of compound23 are added and the mixture is heated for 18 hours at 80° C. A furtherportion of compound 23 (52 mg, 0.14 mmol) is added and the mixture isheated for 24 hours at 80° C. The solution is evaporated to dryness andthe residue is dissolved in 30 ml of CH₂Cl₂ and 10 mL of water. Theaqueous phase is extracted with 4 portions of 30 mL of CH₂Cl₂ and thecombined organic phases are dried over MgSO₄, filtered and thenevaporated to dryness. The residue is purified by chromatography onalumina containing 10% water with a mixture of CH₂Cl₂/MeOH (100/0 to99.3/0.7) as eluent. 46 mg (0.06 mmol) of compound 24 are obtained(corresponding to a yield of 37%), which product has the followingcharacteristics:

R_(f)=0.16, Al₂O₃, CH₂Cl₂/MeOH (98/2).

¹H-NMR (CDCl₃, 200 MHz): δ 2.17-2.28 (m, 2H), 2.61 (t, 2H, ³J=7.5 Hz),3.44 (s, 3H), 3.71 (t, 1H, ³J=7.5 Hz), 3.83 (s, 3H), 4.06-4.19 (m, 12H),4.45-4.70 (m, 4H), 7.82 (d, 2H, ³J=9.0 Hz), 7.86 (s, 2H), 8.13 (d, 2H,³J=8.0 Hz), 8.18 (d, 2H, ³J=9.0 Hz), 8.24 (d, 2H, ³J=8.5 Hz)

Preparation of Compound 25.3HCl

46 mg (0.06 mmol) of 24 and 10 mg (0.25 mmol) of NaOH are dissolved in amixture of 9 mL of MeOH and 3 mL of water in a 50 mL round-bottomedflask. The mixture is heated at 75° C. for 21 hours. The solvents areevaporated off under reduced pressure, the solid is dissolved in 5 mL ofwater and the solution obtained is filtered through Celite. The mediumis acidified with dilute hydrochloric acid solution and the solution isevaporated to dryness. The residue is washed with 2 portions of 2 mL ofwater. 19 mg (0.02 mmol) of 25.3HCl hydrochloride are obtained(corresponding to a yield of 39%) in the form of an orange-yellowpowder, the characteristics of which are as follows:

¹H-NMR (CD₃OD, 200 MHz): δ 2.38-2.52 (m, 2H), 2.84 (t, 2H, ³J=7.0 Hz),4.17 (t, 1H, ³J=7.5 Hz), 4.35 (s, 6H), 4.81-4.85 (m, 4H), 7.87 (d, 2H,³J−9.5 Hz), 7.89 (s, 2H), 8.16 (d, 2H, ³J=9.0 Hz), 8.18 (d, 2H, ³J=8.5Hz), 8.65 (d, 2H, ³J=9.0 Hz).

Example 19 Preparation of Compound 26 of Formula [Eu.(25-4H).H₂O]Na

19 mg of 25.3HCl (24 μmol) are dissolved in a mixture of 15 mL of MeOHand 15 mL of water in a 50 mL round-bottomed flask. To this solution areadded 10 mg (27 μmol) of EuCl₃.6H₂O dissolved in 2.5 mL of MeOH and 2.5mL of water. The solution is heated at 70° C. for one hour. Aftercooling, the pH of the solution is raised to 7.0 with a 0.5% solution ofNaOH in water. The solution is concentrated on a rotary evaporator untilslight cloudiness appears, and THF is then added until a precipitateforms. The yellow solid is isolated by centrifugation and then driedunder vacuum to give 7 mg (8 μmol) of compound 26 (corresponding to ayield of 33%), the characteristics of which are as follows:

FAB⁻/MS: 791.2 (30%), 828.2 ([26-H₂O—Na)]⁻, 50%).

Example 20 Marking of an Anti-Digoxigenine Antibody with Complex 9 andCharacterization by Mass Spectrometry

0.5 mg of complex 9 is added to a solution of anti-digoxigenine antibodycontaining 1.0 mg of antibody dissolved in 500 μL of buffer solution (50mM borate buffer, pH=7.0), corresponding to a 9/antibody ratio of 30:1.The solution is stirred at room temperature for 24 hours, and the markedantibody is then purified according to the procedure described inExample 5 and stored at 4° C.

Characterization by MALDI-TOF mass spectrometry is performed accordingto the procedure described in Example 5, leading to a mass of 49 220 Dafor the marked antibody (47 880 Da for the free antibody), i.e. a degreeof grafting of 2.5.

1. A compound corresponding to formula (I)

in which R¹ is a functional group capable of reacting with the functionspresent on proteins, antibodies or on mineral or organic materials; Xrepresents a single bond or a hydrocarbon-based chain consisting of atleast one group chosen from alkylene groups and alkenylene groupsoptionally comprising at least one hetero atom, and from arylene groups;R² is a group A² that is anionic at neutral pH or an alkylene oralkenylene group containing from 1 to 4 carbon atoms and bearing atleast one such group A², said alkylene or alkenylene group optionallycomprising at least one hetero atom in the chain; R³ represents H or analkylene or alkenylene group containing from 1 to 5 carbon atoms andoptionally containing at least one hetero atom in the chain, said groupoptionally bearing at least one group A³ that is anionic at neutral pH;R⁴ is chosen from the groups corresponding to the formula—(C)_(n)—C—Z¹—C—C—Z²—C—A⁴ in which n is equal to 1 or 2, Z¹ and Z²represent, independently of each other, a hetero atom chosen from O andN, at least one being a nitrogen atom forming part of an aromaticheterocycle with the two carbon atoms surrounding it, and A⁴ is a groupthat is anionic at neutral pH, in which the atom bearing the anioniccharge is in the γ position relative to Z²; R⁵ is chosen from the groupsdefined for R⁴ or from groups corresponding to the formula—C—C—E¹—C—C—E²—C—A⁵ in which E¹ and E² represent, independently of eachother, a hetero atom chosen from O and N, and A⁵ is a group that isanionic at neutral pH, in which the atom bearing the anionic charge isin the γ position relative to E².
 2. The compound as claimed in claim 1,wherein the substituent R¹ is selected from the group consisting ofamino, thio, cyano, isocyano, acridinyl, hydrazino, haloacetate,anhydride, triazo, carbonyl, nitrobenzoyl, sulfonyl, thionyl, halide,epoxide, aldehyde, imidazole, hydroxyphenyl, mercapto, N-succinimidylester, N-sulfosuccinimidyl ester, maleimido, hydroxyl, carboxyl,thiocyano, and isothiocyano groups.
 3. The compound as claimed in claim1, wherein the substituent R³ is a group A² that is anionic at neutralpH.
 4. The compound as claimed in claim 1, wherein the substituent R³ isH or a C₁ to C₃ alkyl.
 5. The compound as claimed in claim 1, whereinthe groups Z¹ and Z² of R⁴ form part of an aromatic heterocyclic group.6. The compound as claimed in claim 1, wherein n is equal to
 1. 7. Thecompound as claimed in claim 1, wherein one of the segments —C—Z¹—C— or—C—Z²—C— forms part of a heterocyclic group chosen from pyridyl,pyrimidinyl, quinolyl and isoquinolyl groups.
 8. The compound as claimedin claim 1, wherein the segment —C—Z¹—C—C—Z²—C— is selected from thegroup consisting of 2,2′-bipyridinyl, 1,10-phenanthrolinyl,2,2′-bisquinolyl, 2,2′-bisisoquinolyl and 2,2′-bipyrimidinyl groups,said groups optionally bearing alkyl or alkoxy substituents on at leastone carbon atom of a heterocycle.
 9. The compound as claimed in claim 1,wherein R⁵ selected from the group consisting of:

in which R⁶ and R⁷ represent alkyl chains containing from 1 to 5 carbonatoms and optionally containing one or more hetero atoms.
 10. Thecompound as claimed in claim 1, wherein R⁴ and R⁵ are identical.
 11. Thecompound as claimed in claim 1, wherein the groups A², A³, A⁴ and A⁵that are anionic at neutral pH are chosen, independently of each other,from —CO₂H, —SO₃H, —P(O)(OR)OH, —P(O)R(OH) and —P(O)(OH)₂ groups inwhich R is an alkyl group or an aryl group.
 12. The compound as claimedin claim 1, wherein the compound is in cationic form, the nitrogenbearing the substituents R⁴ and R⁵, and optionally the hetero atoms Z¹,Z², E¹ and E², being in protonated form.
 13. The compound as claimed inclaim 1, wherein the compound is in anionic form, the various groups A¹being in the form of salts.
 14. The compound as claimed in claim 1,wherein the compound is in zwitterionic form, the nitrogen bearing thesubstituents R⁴ and R¹, and optionally the hetero atoms Z¹, Z², E¹ andE², being in protonated form, and the various groups A^(i) being in theform of salts.
 15. The compound as claimed in claim 1, wherein X is anarylene group comprising one or more fused or unfused aromatic nuclei,said nucleus (nuclei) optionally bearing one or more aliphatichydrocarbon-based groups.
 16. The compound as claimed in claim 1,wherein the group X is an alkylene or alkenylene group containing from 1to 10 carbon atoms.
 17. The compound as claimed in claim 1, wherein thegroup X is an arylene group containing from 5 to 10 carbon atoms.